ABSTRACT Ebola virus (EBOV) is classified as a filamentous, enveloped, non-segmented negative-sense RNA carrying Category A Priority Pathogen by the NIH and CDC. Infections with EBOV cause severe hemorrhagic fever and high mortality in humans and non-human primates (NHPs). During the Ebola outbreak of late 2014 in West Africa, the lack of laboratory tests for early Ebola-virus-disease (EVD) detection (preferably before the onset of symptoms) and lack of effective treatments limited the containment and management of the eventual epidemic. To address this critical need for early EVD diagnosis, we have recently customized a platform we developed to enhance biomarker detection to allow sensitive and specific EBOV biomarker quantification in serum samples. Our method uses antibody-modified porous silicon nanodisks (pSiNDs) and high-throughput, high-resolution mass spectrometry (MS) ? together referred to as pSiND-MS ? to greatly enhance the specificity and sensitivity of target peptide detection from complex protein samples. In our customized EVD assay, these pSiNDs are functionalized with custom antibodies to specific peptides of the EBOV matrix protein VP40 in order to efficiently capture these peptides from non-enzymatically-digested serum samples of EBOV-infected hosts. Notably, the starting non-enzymatic digestion step of this assay is used to both inactivate live EBOV particles and to site- specifically digest all serum proteins for subsequent pSiND-MS analysis. We have observed that VP40 peptides are increased in the plasma of infected animals even before EBOV RNA is detectable with currently available diagnostic assays. We have identified EBOV- and EBOV-species-specific VP40 peptides, and hypothesize that multiplex detection of our two selected VP40 peptides will robustly indicate EVD, EBOV species and EBOV load in potentially-infected individuals. We will thus apply pSiND-MS to quantify low-abundance VP40 peptides for early diagnosis of EBOV using a rapid, single-test assay, where we propose to: 1) optimize the pSiND-MS platform for detection of these biomarkers and 2) apply pSiND-MS to longitudinally quantify VP40 levels in sera from EBOV-infected non-human primates at the U.S. Army Medical Research Institute of Infectious Diseases, to produce an informative preclinical EBOV-detection profile. Major advantages of our approach include the simplicity and safety of sample preparation; the use of matrix- assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS equipment found in most hospitals and laboratories (and future use of simplified, miniaturized MS systems); the potential to read the assay on MS/MS instruments for marker sequence confirmation; the specificity and sensitivity of biomarker detection; the use of rapidly-customizable, modular multiplex assay; and rapid diagnosis (2hrs from sample). These characteristics are ideal for a point-of-need application. Successful completion of this proposal will provide a novel technology that can overcome significant hurdles of current diagnostic methodologies for Ebola and other infectious agents, potentially transforming the protocols for care, treatment, and containment of future epidemics.